This grant proposal is designed to study novel cellular and molecular mechanisms of vascular dysfunction in obesity with insulin resistance, two major components of the metabolic syndrome. Specifically, we will study the role that the calcium-dependent protease calpain plays in the inflammatory signaling of angiotensin II (AngII), and in the tissue protective action of AngII receptor blocking therapy in vivo. Our main hypothesis is that in states of obesity-associated insulin resistance, elevated AngII signaling synergizes with neutrophil-derived myeloperoxidase (MPO) to cause vascular dysfunction via activation of calpain in the vascular endothelium. The results of the integrative studies proposed here may uncover novel mechanisms of vascular complications in the metabolic syndrome. They will also provide an in depth understanding of the pleiotropic actions of AngII receptor blocking therapy in such disease state. Evidence has been accumulating in the literature to indicate that AngII, the key effector of the renin- angiotensin system (RAS), induces vascular inflammation, and that obesity-associated insulin resistance upregulates the RAS and AngII signaling. Despite intense research, the cellular and molecular mechanisms of the inflammatory action of AngII remain largely unknown, which limits therapeutic interventions in the ever-growing obese, insulin resistant U.S. population. Recently, our laboratory has found evidence of increased calpain activity with endothelial dysfunction and leukocyte activation in obese, insulin resistant laboratory rodents. Others have now linked the calpain system to the metabolic syndrome in humans. Preliminary data provided in the present application demonstrate a role for calpain in the inflammatory signaling of AngII, which gives rise to several, unresolved scientific questions. The answering of such questions will ultimately allow us to develop new, effective therapeutic tools to fight vascular disease in humans. To implement this research we will use the Zucker Obese rat, a relevant animal model of obesity-associated insulin resistance, and selected knockout mouse technology. In vivo studies will clarify whether (1) inhibition of AngII signaling prevents calpain activation and actions in the vascular endothelium;(2) circulating neutrophils are mechanistically important to sustain endothelial calpain activation in response to AngII;(3) neutrophil-derived MPO is a key molecular determinant of the AngII/calpain inflammatory signaling cascade;(4) I?B/NF?B plays a mechanistic role in the endothelial dysfunction of calpain. We will utilize the following biochemistry and physiology techniques: western blot analysis, immunohistochemistry and immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, antisense DNA technology, cells and tissue isolation techniques, intravital microscopy, in vivo measurements of nitric oxide and superoxide. We expect that the results of our research will improve the life quality and expectancy of the obese, insulin resistant U.S. population, in addition to lowering the overall economic cost of managing cardiovascular complications. The research proposed in this application intends to provide new biological markers and therapeutic strategies to fight the vascular complications of obesity and insulin resistance. According to the American Heart Association, cardiovascular disease (CDV) remains the No. 1 killer in the United States. The estimated direct and indirect cost of CVD for 2006 is $403.1 billion. We have identified a novel signaling pathway, the calpain system, which helps explain a) how angiotensin II damage the vasculature of body organs, and b) why medications that block the renin-angiotensin system protect the cardiovascular system. We expect that the results of our research will improve life quality and expectancy of the obese, insulin resistant U.S. population, in addition to lowering the overall economic cost of managing cardiovascular disease.